Project Summary All eukaryotes use phosphorylation-based signaling networks, composed of protein kinases and phosphatases, to regulate cellular processes. While global information about kinase signaling has exploded, knowledge of phosphatases has lagged behind. The lack of systematic, unbiased approaches to analyze phosphatase signaling leaves a major gap in our understanding of cellular regulatory networks. For >25 years, my research on calcineurin, the conserved Ca2+/calmodulin-regulated phosphatase, in S. cerevisiae and humans has directly addressed this issue. Calcineurin is ubiquitously expressed and plays critical regulatory roles in the cardiovascular, nervous and immune systems (1); however, only ~70 proteins are currently established as calcineurin substrates (2). Calcineurin dephosphorylates the Nuclear Factor of Activated T-cells (NFAT) transcription factors to activate the adaptive immune response (3), and calcineurin inhibitors, FK506 and cyclosporin A, are commonly prescribed as immunosuppressants (4). However, these drugs cause unwanted effects, including hypertension, diabetes, and seizures by inhibiting calcineurin in non-immune tissues (5), highlighting the need to map human calcineurin signaling pathways systematically. My work elucidates calcineurin signaling through novel approaches based on the discovery of short linear motifs (SLiMs): short degenerate peptide sequences found within regions of intrinsic disorder that determine specific, low- affinity interactions that are essential for signaling (6). Using experimental and in silico SLiM-based methods, we recently defined the human calcineurin signaling network (2). This work uncovered conserved regulation of nuclear pore complexes by calcineurin and showed unexpected calcineurin proximity to centrosomes (2). Future efforts will elucidate Ca2+ and calcineurin signaling at these compartments using a combination of methods that include phosphoproteomics and proximity labeling with faster-labeling biotin ligases (miniTurbo (33), Split TurboID). Fluorescent sensors will be used to probe Ca2+ signals at these locations in vivo. Calcineurin functions at membranes will be analyzed by focusing on CNb1, a little-studied isoform with distinct localization and regulation conferred by its unique, lipidated C-terminal sequences (10). Regulation of CNb1 activity via dynamic palmitoylation will be examined, and CNb1-specific signaling pathways, including regulation of PI4P synthesis, will be established to achieve a comprehensive and mechanistic understanding of this enzyme. Finally, the novel technology, MRBLE-PEP (Microspheres Ratiometric Barcode Lanthenide Encoding coupled to PEPtides) (11), will be developed for quantitative analysis and discovery of SLiMs. Overall this research aims to map human calcineurin signaling pathways systematically and to advance our understanding of SLiM-based signaling more broadly.